Papillomaviruses (PVs) infect the epithelia of animals and man, where they generally induce benign proliferation at the site of infection. In addition, persistent infection by some human papillomaviruses (HPV), especially HPV16, can induce malignant progression of human genital lesions and certain human papillomavirus (HPV) types. Our research is primarily concerned with development of vaccines and other strategies to inhibit HPV infection, as well as elucidation of the papillomavirus life cycle. We previously developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, i.e. pseudovirions. We have exploited this technology in our basic virologic and translational research efforts, leading to development of the first cervicovaginal challenge model for HPVs. Both in the mouse model and in an analogous model developed for the rhesus macaque, infection is prevented by local application of carrageenan, which is an algal polysaccharide widely used in processed food and cosmetics and is the main gelling agent in some over-the-counter lubricants. Two clinical trials of carrageenan as a microbicide to prevent genital HPV infection in young women will begin soon, and we will collaborate on technical aspects of the trials. The pseudovirus-based mouse cerviovaginal challenge model is also being used to explore basic features of papillomavirus infection and the mechanisms of antibody-mediated neutralization of the virus in the female reproductive tract. We believe these studies provide the most detailed mechanistic understanding of how a virus, in a living mammalian host, infects its relevant tissue and how vaccine-induced neutralizing antibodies prevent infection in vivo. Using the mouse model, we determined that transient disruption of epithelial integrity is required for HPV infection, that the first step in the virus life cycle involves binding of the virus to the basement membrane, and that the virus transfers to target epithelial cells only after the L2 protein in the capsid has undergone an enzymatic cleavage while on the basement membrane. The cleavage of L2 is associated with a conformational change in the capsid, leading to exposure of highly conserved L2 cross-neutralization epitopes that lie C-terminal to the cleavage site. In a process that takes several hours, the virions transfer from the basement membrane to the surface of keratinocytes invading the site of trauma, and the virions are then internalized. We have determined that antibodies induced by L1 VLP vaccines (which are now FDA-approved) and L2 vaccines (which we are developing as a more broadly cross type-neutralizing alternative) block in vivo infection by distinct mechanisms. At high concentrations, L1 VLP-induced antibodies block virion binding to the basement membrane, while at low concentrations, they permit basement membrane binding but prevent binding to target keratinocytes. Since the L2 cross-neutralization epitopes are cryptic, and are not exposed until after the virions have been bound to the basement membrane, L2 antibodies that broadly cross-neutralize HPV types do not interfere with initial basement membrane binding. Once the L2 epitopes are exposed, while the virions remain on the basement membrane, the L2 antibodies can bind to the virions, and abrogate the infectious process by preventing transfer of the virions to target keratinocytes. Using HPV pseudoviruses, we previously developed a high throughput in vitro HPV neutralization assay that has become the gold standard for the field. In the course of the L2 vaccine studies described above, it became clear that the assay was an insensitive measure of neutralizing antibodies induced by candidate L2 vaccines, as passive transfer of immune serum at concentrations that protected mice from HPV challenge were negative in the in vitro neutralization assay. Based on the improved understanding of the in vivo infectious process and the mechanism by which L2 vaccines conferred protection, we developed a new in vitro neutralizing assay that is approximately three orders of magnitude more sensitive than the standard assay in detecting L2 neutralizing antibodies. Using this assay, sera that are protective in the mouse challenge model but are negative with the standard in vitro assay have been found to contain neutralizing activity in the new in vitro assay. The new assay should have considerable utility for determining the immunogenicity of candidate L2 vaccines and for clinical trials of L2 vaccines. Our development of a method to induce efficient HPV pseudovirus infection of the female genital tract after transient epithelial disruption with nonoxynol-9 (N-9) has proven to be the key to our recent development of an effective intravaginal vaccination strategy. In patent pending studies, we have found that intravaginal pseudovirus vaccination of N-9-treated mice induces strong systemic and mucosal T and B cell responses to target antigens transduced by the pseudovirions. Systemic responses rival those induced by previously optimized Ad5 vectors. Intravaginal responses are remarkably strong, with up to 80% of all intravaginal CD8 T cells staining tetramer positive for the targeted antigen. Most of the induced T cells appear to be intraepithelial and are maintained in the vaginal tract at least 100 days after vaccination. Intravaginal pseudovirus vaccination is a promising approach for focusing immune responses to the female genital tract that may increase the effectiveness of therapeutic vaccines directed against genital HPV infection or herpes simplex virus infection.